Reduced creatine-stimulated respiration in doxorubicin challenged mitochondria: particular sensitivity of the heart.
ABSTRACT Doxorubicin (DXR) belongs to the most efficient anticancer drugs. However, its use is limited by a risk of cardiotoxicity, which is not completely understood. Recently, we have shown that DXR impairs essential properties of purified mitochondrial creatine kinase (MtCK), with cardiac isoenzyme (sMtCK) being particularly sensitive. In this study we assessed the effects of DXR on respiration of isolated structurally and functionally intact heart mitochondria, containing sMtCK, in the presence and absence of externally added creatine (Cr), and compared these effects with the response of brain mitochondria expressing uMtCK, the ubiquitous, non-muscle MtCK isoenzyme. DXR impaired respiration of isolated heart mitochondria already after short-term exposure (minutes), affecting both ADP- and Cr-stimulated respiration. During a first short time span (minutes to 1 h), detachment of MtCK from membranes occurred, while a decrease of MtCK activity related to oxidative damage was only observed after longer exposure (several hours). The early inhibition of Cr-stimulated respiration, in addition to impairment of components of the respiratory chain involves a partial disturbance of functional coupling between MtCK and ANT, likely due to interaction of DXR with cardiolipin leading to competitive inhibition of MtCK/membrane binding. The relevance of these findings for the regulation of mitochondrial energy production in the heart, as well as the obvious differences of DXR action in the heart as compared to brain tissue, is discussed.
- SourceAvailable from: Ben Loos[Show abstract] [Hide abstract]
ABSTRACT: Anthracyclines, such as doxorubicin, are among the most valuable treatments for various cancers, but their clinical use is limited due to detrimental side-effects such as cardiotoxicity. Doxorubicin-induced cardiotoxicity is emerging as a critical issue among cancer survivors and is an area of much significance to the field of cardio-oncology. Abnormalities in mitochondrial functions such as defects in the respiratory chain, decreased adenosine triphosphate production, mitochondrial DNA damage, modulation of mitochondrial sirtuin activity and free radical formation have all been suggested as the primary causative factors in the pathogenesis of doxorubicin-induced cardiotoxicity. Melatonin is a potent anti-oxidant, is non-toxic and has been shown to influence mitochondrial homeostasis and function. Although a number of studies support the mitochondrial protective role of melatonin, the exact mechanisms by which melatonin confers mitochondrial protection in the context of doxorubicin-induced cardiotoxicity remain to be elucidated. This review focuses on the role of melatonin on doxorubicin-induced bioenergetics failure, free radical generation and cell death. A further aim is to highlight other mitochondrial parameters such as mitophagy, autophagy, mitochondrial fission and fusion and mitochondrial sirtuin activity which lack evidence to support the role of melatonin in the context of cardiotoxicity.This article is protected by copyright. All rights reserved.Journal of Pineal Research 09/2014; · 7.81 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Historically, cellular trafficking of lipids has received much less attention than protein trafficking, mostly because its biological importance was underestimated, involved sorting and translocation mechanisms were not known, and analytical tools were limiting. This has changed during the last decade, and we discuss here some progress made in respect to mitochondria and the trafficking of phospholipids, in particular cardiolipin. Different membrane contact site or junction complexes and putative lipid transfer proteins for intra- and intermembrane lipid translocation have been described, involving mitochondrial inner and outer membrane, and the adjacent membranes of the endoplasmic reticulum. An image emerges how cardiolipin precursors, remodeling intermediates, mature cardiolipin and its oxidation products could migrate between membranes, and how this trafficking is involved in cardiolipin biosynthesis and cell signaling events. Particular emphasis in this review is given to mitochondrial nucleoside diphosphate kinase D and mitochondrial creatine kinases, which emerge to have roles in both, membrane junction formation and lipid transfer.Chemistry and Physics of Lipids 12/2013; · 2.59 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Heart failure is a common complication of doxorubicin (DOX) therapy. Previous studies have shown that DOX adversely impacts cardiac energy metabolism, and the ensuing energy deficiencies antedate clinical manifestations of cardiac toxicity. Brief exposure of cultured cardiomyocytes to DOX significantly decreases creatine transport, which is the cell's sole source of creatine. We present the results of a study performed to determine if physiological creatine supplementation (5 mmol/L) could protect cardiomyocytes in culture from cellular injury resulting from exposure to therapeutic levels of DOX. Creatine supplementation significantly decreased cytotoxicity, apoptosis, and reactive oxygen species production caused by DOX. The protective effect was specific to creatine and depended on its transport into the cell.Cardiovascular Toxicology 09/2014; · 2.06 Impact Factor
Reduced creatine-stimulated respiration in doxorubicin challenged
mitochondria: Particular sensitivity of the heart
Malgorzata Tokarska-Schlattnera,d,⁎, Max Dolderb, Isabelle Gerbera, Oliver Speerc,
Theo Wallimanna, Uwe Schlattnera,d
aInstitute of Cell Biology, ETH Zurich, CH-8093 Zurich, Switzerland
bInstitute of Medical Microbiology, University of Zurich, CH-8006 Zurich, Switzerland
cEC-Laboratory, Children Hospital, University of Zurich, CH-8032 Zurich, Switzerland
dINSERM, U884, Laboratory of Fundamental and Applied Bioenergetics, University Joseph Fourier, F-38041 Grenoble, France
Received 7 December 2006; received in revised form 19 August 2007; accepted 23 August 2007
Available online 8 September 2007
Doxorubicin (DXR) belongs to the most efficient anticancer drugs. However, its use is limited by a risk of cardiotoxicity, which is not
completely understood. Recently, we have shown that DXR impairs essential properties of purified mitochondrial creatine kinase (MtCK), with
cardiac isoenzyme (sMtCK) being particularly sensitive. In this study we assessed the effects of DXR on respiration of isolated structurally and
functionally intact heart mitochondria, containing sMtCK, in the presence and absence of externally added creatine (Cr), and compared these
effects with the response of brain mitochondria expressing uMtCK, the ubiquitous, non-muscle MtCK isoenzyme. DXR impaired respiration of
isolated heart mitochondria already after short-term exposure (minutes), affecting both ADP- and Cr-stimulated respiration. During a first short
time span (minutes to 1 h), detachment of MtCK from membranes occurred, while a decrease of MtCK activity related to oxidative damage was
only observed after longer exposure (several hours). The early inhibition of Cr-stimulated respiration, in addition to impairment of components of
the respiratory chain involves a partial disturbance of functional coupling between MtCK and ANT, likely due to interaction of DXR with
cardiolipin leading to competitive inhibition of MtCK/membrane binding. The relevance of these findings for the regulation of mitochondrial
energy production in the heart, as well as the obvious differences of DXR action in the heart as compared to brain tissue, is discussed.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Anthracycline; Creatine kinase; Cardiotoxicity; Isolated mitochondria; Creatine-simulated respiration
Doxorubicin (DXR) and other anthracyclines are among the
most potent chemotherapeutic drugs for the treatment of variety
of human cancers. Cardiotoxic side effects represent, however,
a serious constraint for long-term DXR chemotherapy. The
molecular mechanism(s) responsible for this tissue-specific
DXR-toxicity in the heart are still unclear, but molecular
damage by DXR has been ascribed to oxidative stress. Cardiac
injury has been related to the impairment of the essential
components of myocardial energy metabolism, including the
mitochondrial generation of high-energy phosphates, which
provides more than 90% of the ATP used by cardiomyocytes
(for a review, see ).
to permanent, high and fluctuating demand is achieved by
several means, among others by a high content of mitochondria,
high energy phosphate transfer systems [2–5]. Mitochondrial
organization contributes to the functioning of these systems via
metabolic channeling and control of mitochondrial respiration
by mitochondrial creatine kinase (MtCK), acting as a powerful
modulator/amplifier for the regulatory action of ADP. As we
could demonstrate in vitro , anthracyclines show multiple
interference with MtCK, impairing several functional properties
of the enzyme.
Available online at www.sciencedirect.com
Biochimica et Biophysica Acta 1767 (2007) 1276–1284
⁎Corresponding author. Laboratoire de Bioenergetique Fondamentale et
Appliquee, INSERM U884 Universite Joseph Fourier, 2280 rue de la Piscine, F-
38041 Grenoble, France. Tel.: +33 476 63 53 99; fax: +33 4 76 51 42 18.
E-mail address: firstname.lastname@example.org
0005-2728/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
The mitochondrial isoenzymes of CK, sarcomeric sMtCK,
expressed in striated muscles including the heart, and
ubiquitous uMtCK found in many other organs, mainly in
the brain, are strictly localized within the cristae, as well as in
the intermembrane space of mitochondria. MtCK is a
peripheral membrane protein and binds to the outer leaflet of
the inner mitochondrial membrane, interacting specifically with
cardiolipin and even inducing the formation of specific
cardiolipin membrane domains . MtCK is enriched in the
so-called contact sites between inner and outer membrane,
where it forms functional microcompartments together with the
transmembrane proteins adenine nucleotide translocator (ANT)
in the inner membrane and mitochondrial porin (VDAC,
voltage-dependent anion channel) in the outer membrane
(reviewed in [4,8–10]). By reversible transfer of phosphoryl
groups between ATP and Cr, MtCK catalyzes intramitochon-
drial regeneration of ADP and produces easily diffusible “high-
energy” phosphocreatine (PCr). While PCr leaves the mito-
chondria by porin to supply the large cytosolic PCr pool,
regenerated ADP is immediately re-imported by ANT into the
matrix space, thus functionally coupling the MtCK reaction and
mitochondrial ATP synthesis to oxidative phosphorylation.
Repeated recycling of ADP stimulates the action of the
respiratory chain and increases the turnover of adenine
nucleotides. The privileged exchange of MtCK substrates and
products between MtCK, ANT and porin is called functional
coupling [3,4,9,11]. In co-operation with cytosolic CK
associated to ATP-requiring reactions, it links elevated work-
load directly to mitochondrial ATP generation. This system is
not only able to account for rapid and high changes in
workload [2,5] but has additional beneficial functions.
Increased respiratory rate due to intramitochondrial regenera-
tion of ADP can prevent both free radical formation in
energized mitochondria [12,13] and opening of the permeabil-
ity transition pore, a trigger of apoptosis .
During DXR treatment, MtCK is highly exposed to the
drug's toxic action as both share cardiolipin as a common
binding partner in the inner mitochondrial membrane. High
affinity binding of DXR to cardiolipin, shown in numerous
previous studies, leads to selective accumulation of the drug in
mitochondria. Thus, high local concentrations of DXR are
reached in the microenvironment of MtCK, resulting in a
competitive inhibition of MtCK/membrane binding [6,15–17].
As a consequence, MtCK is released from the inner mitochon-
drial membrane. The common location also situates MtCK at
the very origin of the formation of reactive oxygen species
(ROS) by redox cycling of DXR, largely mediated by
mitochondrial oxidoreductive enzymes . Oxidative stress
is of particular danger to CK isoenzymes that have been
recognized as very sensitive to loss of function by oxidation and
In our previous in vitro study with recombinant MtCK
isoenzymes, we have shown that anthracyclines, including DXR,
affect essential properties of recombinant, purified mitochondrial
creatine kinase, such as its membrane binding, oligomeric state
and enzymatic activity . In addition, the cardiac isoenzyme
sMtCK turned out to be more sensitive to the drug than the
that this differential sensitivity, together with high oxidative
capacity of the heart and high cardiolipin content of cardiac
mitochondria, could contribute to the cardiac specificity of the
drug's toxic side effects. The aim of the present study was
therefore to assess the effect of DXR on MtCK in its natural
environment, inside the intact mitochondrion. First, we assessed
Cr-stimulated respiration in isolated functionally intact heart
mitochondria. There is some evidence that functional coupling is
impaired by DXR , but the potential effects of DXR on Cr-
stimulated respiration have never been investigated in detail with
clinically relevant drug concentrations. Furthermore, we have
compared the response of heart and brain mitochondria to such
clinically relevant DXR concentrations, thus aiming at a
comparison of the sMtCK and uMtCK isoforms. Finally, the
kinetics of mitochondrial respiration in response to submaximal
ADP concentration and in the presence or absence of physiolog-
that the stimulatory effect of Cr on respiration, due to metabolite
channeling within the ANT-MtCK-porin complex, was rapidly
abolished by DXR, an effect that was particularly pronounced in
heart mitochondria. This study clearly points to Cr-stimulated
respiration as an important process to be taken into account when
evaluating cardiac side effects of DXR.
2. Materials and methods
2.1. Preparation of mitochondria
Mitochondria were isolated by differential centrifugation according to
Dolder et al.  and subsequently purified by Percoll gradient. The whole
procedure was performed at 4 °C. Briefly, bovine hearts were supplied by
slaughter house, rat hearts and brains were freshly removed from laboratory
animals (Wistar rats). Organs were placed in ice-cold isolation buffer (225
mannitol, 75 mM sucrose, 10 mM HEPES, pH 7.4) supplemented with 1 mM
EDTA and 0.1% bovine serum albumin, cut with surgical scissors, and
homogenized using Polytron (PT 3000, Kinematica AG) and/or Teflon potter
(for brain only a Potter homogenizor was used). Homogenate (H) was
centrifuged at 1000×g for 10 min. The resulting supernatant (H1) was
centrifuged at 12,000×g for 10 min. Obtained in this way, the crude
mitochondrial pellet (CM) was further purified by 35-min centrifugation at
100,000×g on a 25% Percoll gradient. Percoll was removed from the purified
mitochondrial fraction (PM) by washing (centrifugation at 12,000×g for 10 min)
twice in isolation buffer. Mitochondrial protein concentration was measured
using the Bio-Rad Protein Assay with bovine serum albumin as a standard.
Purity and structural integrity of mitochondrial preparation was checked by
Western blotting and electron microscopy, respectively.
Doxorubicin hydrochloride was purchased by Fluka (Buchs, Switzerland);
10 mM stock solution was prepared as described by . The disodium salts of
ADP and NADP were obtained from Roche, PCr from Calbiochem, and P1P5-di
(adenosine-5′)-pentaphosphate pentasodium salt from Sigma. All other chemi-
cals were at least of reagent grade.
2.3. Incubation of mitochondria with doxorubicin
If not stated otherwise, mitochondria were resuspended in isolation buffer to
the desired concentration (0.5–1 mg/ml) and incubated with doxororubicin 1 h
at room temperature (RT) with constant shaking. Doxorubicin dose used has
been always adjusted to mitochondrial protein concentration.
1277M. Tokarska-Schlattner et al. / Biochimica et Biophysica Acta 1767 (2007) 1276–1284
2.4. Respiratory measurements
Mitochondrial oxygen consumption was measured at 25 °C with a high
performance OROBOROS oxygraph (Anton Park, Innsbruck, Austria) in
respiration buffer (250 mM sucrose, 10 mM Tris/Mops, pH 7.4) supplemented
with 10 mM Pi, 2 MgCl2, 2 μM rotenone. ADP- and Cr-simulated respiration
was analyzed in a setup described by Jacobus and Lehninger (; see also )
with slight modifications. Resting respiration (State 4) was induced by 5 mM
succinate(10mM for brain),andsubsequentlystimulated by25 μM/1 mMADP.
Stimulation of respiration by ADP was analyzed in the absence and in the
presence of 10 mM Cr. At the end of the measurement cycle, ANTwas blocked
by addition of atractyloside (40 μM) and the level of resting respiration was
checked. Alternatively, uncoupler (1μM carbonyl cyanide m-chlorophenylhy-
drazone; CCCP)-stimulated respiration was analyzed as an index of maximal
electron transport capacity.
2.5. Enzymatic activity measurements
The reverse CK reaction (ATP production) was measured in a coupled
photometric enzymatic assay as described earlier , with all reagents prepared
without addition of reducing agent (β-mercaptoethanol) in order to not
counteract the effects of DXR (see ). Values were corrected for other ADP-
consumingreactions(e.g., by adenylate kinase) by subtractingactivity measured
in parallel in the absence of PCr, the CK-specific substrate. One International
Unit of CK activity (1 U) corresponds to 1 μM of PCr transphosphorylated per
min at pH 7.0 and 25 °C.
2.6. Cellulose polyacetate electrophoresis (CPAE)
CK isoenzymes were separated by native CPAE for 1 h at 150 V at room
temperature . Subsequently, the CK bands were visualized by an enzymatic
color reaction coupled to the CK reverse reaction; the reaction mixture was
supplemented with 100 μM P1P5-di(adenosine-5′)-pentaphosphate to inhibit
interfering adenylate kinase activity.
2.7. Binding/detachment assay
Frozen in liquid N2 and thawed mitochondria were centrifugated at
13,000 rpm for 5 min and MtCK was probed in supernatant by immunoblotting.
2.8. SDS-PAGE electrophoresis and immunoblotting
Proteins separated by SDS-PAGE were semi-dry blotted onto nitrocellulose
transfer membranes (Protran, Schleicher and Schuell). After blocking, blots were
immunostained with specific antibodies for 2 h atroomtemperature. The blocking
buffer consisted of 4% milk, TBS, and 0.05% Tween 20. The primary antibodies
were diluted in blocking buffer by the following factors: 1:200 for anti-VDAC1
antibody (Santa Cruz Biotechnology), 1:500 for anti-cytochrome c antibody (BD
Biosciences Pharmingen), 1:1000 for anti-MtCK antibodies (generated and
characterized previously in our laboratory ), anti-SNAP25 (soluble NSF
attachment protein, BD Biosciences Pharmingen) and anti-PSD95 (post-synaptic
and blots were incubated for 1 h at room temperature. Blots were developed with
Chemiluminescent Detection Kit (AppliChem GmbH).
2.9. Statistical analysis
Data are presented as mean±standard deviation (S.D.). Student's t-test was
used to compare two means. P≤0.05 was taken as significant. Experiments
were performed at least three times.
3. Results and discussion
3.1. Mitochondria from the heart and brain
In vitro, muscle-specific sMtCK present in the heart was
found to be more prone to oxidative damage by DXR as
compared to uMtCK expressed in the brain and most other
organs [6,24,30]. To examine DXR effects on the MtCK-Cr
system within the mitochondria of these very organs, a
procedure for their isolation and quality control was established.
Quality of the mitochondrial preparations was reproducibly
high, as judged from immunoblotting (Fig. 1), functional
analysis (respiratory control ratio of ∼5 with succinate as a
substrate), as well as electron microscopy (data not shown).
Both the crude mitochondrial fraction (CM) and the final
Percoll preparations (PM) from the two tissues were highly
enriched in mitochondria. Purification on a Percoll gradient
efficiently eliminated contamination of heart CM by endoplas-
mic reticulum, while in the brain PM, a small contamination by
synaptosomes and cytosolic BCK persisted despite repeated
washing. The mitochondrial preparations from the heart and
brain showed significant amounts of MtCK, but differed in their
Fig. 1. Analysis of purity of mitochondrial preparations. Tissue homogenates (H, H1; prepared as described in Materials and methods), crude mitochondria (CM) and
Percoll purified mitochondria (PM) from rat heart and brain were probed by immunoblotting for selected mitochondrial markers (cytochrome c, VDAC, MtCK),
markers for possible contaminating organelles (calreticulin for endoplasmic reticulum, SNAP25 and PSD95 for synaptosomes), as well as for the presence of a
cytosolic isoform of brain-type creatine kinase (BCK). Samples were loaded on the SDS-PAGE gels either in duplicates (each 10 μg) or when marked with asterisk in
two different amounts (10 and 25 μg protein).
1278 M. Tokarska-Schlattner et al. / Biochimica et Biophysica Acta 1767 (2007) 1276–1284
specific activities for this kinase (Fig. 2A). It has to be
mentioned that also the distribution of CK activity between
mitochondrial and cytosolic isoenzymes is different in these
organs (Fig. 2B).
3.2. Doxorubicin affects ADP-stimulated respiration in isolated
intact heart mitochondria
Respiratory analysis was performed with succinate as
substrate and rat or bovine heart mitochondria. The latter,
however, remained more stable and well coupled under
conditions of DXR incubation. We first checked the effect of
relatively low DXR concentrations (up to 300 μM DXR/mg
mitochondrial protein) on respiratory states 4 and 3, the latter
initiated by 1 mM ADP. At these DXR concentrations, there
was no rapid effect of the drug on respiratory state 4 (Fig. 3A
for 100 μM DXR). In contrast, an immediate inhibition of
ADP-stimulated state 3 was observed (Fig. 3A, B), and
preincubation of mitochondria with DXR for 1 h at RT
increased this inhibitory effect (Fig. 3C). As seen with control
mitochondria, the latter incubation had a negligible effect on
respiratory control ratio, as compared to incubation on ice.
The magnitude of state 3 inhibition is consistent with earlier
data for pigeon and rat heart mitochondria [31,32]. DXR also
affects uncoupler-stimulated respiration (Fig. 3B, see also
), indicating an impairment of the respiratory chain. Since
our assay supplies electrons to complex II, it is likely that an
inhibition of complex IV is involved in producing this effect
(see  and literature therein). DXR inhibition was consis-
tently higher for uncoupler- as compared to ADP-stimulated
respiration, consistent with general benefits of the coupled
process (see ). Based on these results, in further
experiments mitochondria were incubated for 1 h at RT with
a drug dose not exceeding 100 μM DXR/mg mitochondrial
protein. Such a DXR concentration is relevant since (i)
mitochondria selectively accumulate DXR to concentrations
much higher than plasma levels, the latter reaching up to 2–
6 μM after bolus injection ); (ii) the incubation mixture
containing the mitochondria contains a relatively high protein
concentration, and (iii) it turned out that it is rather the relation
between concentrations of DXR and mitochondrial proteins
that is important for DXR effects in mitochondria (data not
shown and ).
Fig. 2. CK enzymatic activity and isoform pattern in the heart and brain. (A)
Specific activity of ADP-consuming reactions in rat heart and brain mitochondria.
Activity measured in the absence of PCr (white bar), in the presence of PCr
(hatched bar) and difference between activities measured in the presence and
absence of PCr (black bar), representing genuine CK activity. All values were
related to CK activity in rat heart mitochondria, which was 4.8±0.6 U/mg. (B)
Distribution of total CK activity between cytosolic and mitochondrial isoforms in
rat heart and brain, assessed by CPA electrophoresis. Isoforms: dimeric cytosolic
CK—muscle-type MCK and brain-type BCK, forming homodimers (MM-,
BBCK) and heterodimers (MBCK). Dimeric (d) or octameric (o) mitochondrial
CK—sarcomeric (sMtCK) and ubiquitous (uMtCK). Heart expresses mainly
MMCK and sMtCK, but less MBCK, and BBCK. Brain expresses mainly BBCK
and uMtCK, but no MM- or MB-CK.
CCCP (gray trace). Upper panel: oxygen content in the measurement chamber, lower panel: corresponding derivatives, showing velocities of oxygen consumption (respiratory
with 40 μM atractyloside (Atr). Mean absolute values of respiratory rate in states 4 and 3 were the following: 27±5 and 125±23 nmol O2/min mg protein.
1279M. Tokarska-Schlattner et al. / Biochimica et Biophysica Acta 1767 (2007) 1276–1284
3.3. Doxorubicin affects Cr-stimulated respiration in heart
mitochondria before inhibiting sMtCK enzymatic activity
We have compared the effect of a 1-h incubation of heart
mitochondria with DXR on the kinetics of mitochondrial
respiration in response to submaximal ADP concentrations
(respiratory rate between states 4 and 3) in the presence and
absence of Cr (Fig. 4A). The stimulatory effect of Cr at these
ADP concentrations is the most significant in normally
functioning mitochondria . In the absence of Cr, energized
mitochondria (with excess of succinate and inorganic phos-
phate) become already activated upon addition of submaximal
ADP concentrations. The rate of oxygen consumption rapidly
increases, but the respiratory burst is terminated as soon as all
ADP is phosphorylated. DXR showed an effect on ADP stim-
ulation as observed before.
In the presence of Cr, external addition of ADP leads to an
immediate strong stimulation (transitory phase, Fig. 4A),
followed by a prolonged activation (near steady-state phase,
see arrows in Fig. 4A, lower panel). It should be noted that the
transitory phase seen after ADP addition in the presence of Cr is
a genuine event observed also elsewhere . It does not occur
after addition of the equivalent amount of solvent, and its
amplitude correlates with the amount of ADP added, not with
added volume. As verified by immunoblotting (Fig. 1A) and
CPA electrophoresis (data not shown), there is no other
contaminating CK isoform present in the cardiac mitochondrial
preparations. Thus, prolonged activation corresponds to
intramitochondrial ADP regeneration by the MtCK reaction,
using mitochondrially produced ATP and the added Cr, as it has
been shown for heart mitochondria in numerous earlier studies
(for state-of-the-art reviews, see ). Such local channeling is
due to the formation of MtCK-ANT microcompartments and
the resulting high affinity of ANT for MtCK-produced ADP
[4,11,14]. The experimental setup used here does not allow for
exact determination of Km(ADP) as done elsewhere , since
externally added ADP is consumed, leading to decreasing ADP
concentrations and complex respiration kinetics. In contrast, the
setup avoids complex additives to the incubation medium, ADP
generation by endogenous ATPases or ADP regenerating sys-
tems like hexokinase, which may all interfere themselves with
DXR and thus complicate the analysis of drug effects (data not
shown). However, this direct approach allows analysis of DXR
effects that are exclusively revealed by the presence of ADP and
Cr and that are due to endogenous mitochondrial components
like the MtCK microcompartments and the respiratory chain.
With bovine heart mitochondria, DXR inhibited stimulation of
respiration by Cr. When quantified, this effect was present in the
steady-state phase (Fig. 4B), and even more significant in the
transitory phase directly following ADP addition (Fig. 4C).
Fig. 4. Effect of DXR on Cr-stimulated respiration in heart mitochondria. (A) Representative traces of kinetics of oxygen consumption in control and DXR-treated
mitochondria in response to submaximal ADP concentrations (25–100 μM) in the absence and presence of 10 mM Cr (upper panel) and corresponding derivatives,
showing velocities of oxygen consumption (respiratory rates; lower panel). Bovine heart mitochondria at 0.5 mg/ml were preincubated without (control, CTR) or with
50 μM DXR during 1 h at RT. (B, C) Quantification of stimulatory effect of Cr in near steady-state phase (marked with arrow; B) and in transitory phase (peak; C),
calculated as difference of the respiratory rate, v, in the presence and absence of 10 mM Cr.⁎P≤0.05; °P=0.08 (n=3–4). Other experimental details as for Fig. 3.
1280M. Tokarska-Schlattner et al. / Biochimica et Biophysica Acta 1767 (2007) 1276–1284
Similar DXR effects were observed in rat heart mitochondria,
which were, however, consistently less stable at RT (not
The observed inhibition of Cr-stimulated respiration occurs
likely through different mechanisms. First, a more general
impairment of the respiratory chain may play a role, as it oc-
curred already during ADP-stimulated respiration (see above)
and may be even more important at the higher respiratory rates
induced by Cr stimulation. However, the pronounced inhibitory
effect especially under conditions of Cr stimulation rather
indicates a major involvement of the MtCK microcompartment,
mainly a direct impairment of MtCK and its coupling to
ANT. The correct functioning of MtCK largely depends on
enzymatic activity and membrane binding of the octameric
enzyme [3,37–39].We have analyzed the effectof DXRon both
properties (Fig. 5A–C). After 1-h incubation of bovine heart
mitochondria with DXR, i.e., when Cr-stimulated respiration
was already impaired, there was no decrease in MtCK
enzymatic activity (Fig. 5A). A significant decrease of MtCK
activity was only observed after longer exposure (several hours;
Fig. 5B). The protective effect of reduced glutathione (15 mM
GSH; Fig. 5B) indicated an involvement of oxidative damage.
These findings are consistent with our earlier in vitro data with
recombinant enzyme, where enzymatic inactivation of MtCK
by DXR was a rather delayed process and due to oxidation of
essential cysteines . Although redox cycling of DXR would
be more prominent in isolated mitochondria than in MtCK
protein solutions due to numerous potential sites of anthraqui-
none reduction, the data show that MtCK inactivation in this
experimental system is still a relatively slow process. This was
also observed in different other model systems, where in-
activation of MtCK by DXR was not immediate [9,40]. More
pronounced DXR effects were only reported after activation of
the drug with exogenous HRP/H2O2or its coupling with iron
In contrast, the effect of DXR on MtCK membrane binding
similar to our earlier in vitro experiments [6,9]. Detachment of
MtCK from mitochondrial membranes was analyzed by
immunodetection of MtCK released into the supernatant after
a freeze–thawing cycle (Fig. 5C). Already after 1-h incubation
the membranes. Such a solubilization of MtCK may render its
active sites more accessible in the enzymatic assay, thus
explaining the even slight but significant increase in MtCK
activity after 1 h of incubation with DXR (Fig. 5A). However,
detachment of MtCK reduces its coupling to ANTand can thus
play a key role in the inhibition of its function in Cr stimulation
of respiration. It is to note that in all experiments presented here,
mitochondria were incubated with DXR in the absence of
respiratory substrates. If the latter were added concomitantly
with DXR, inactivation and detachment of MtCK from
membranes, as well as oxidation of mitochondrial proteins as
assessed by oxyblot were enhanced (data not shown). However,
in our assay conditions, such a treatment affected substantially
the integrity of mitochondrial membranes (data not shown)
and was thus not convenient for subsequent respiratory
3.4. Differential action of DXR in brain
Under various harmful conditions, including DXR exposure,
the muscle-specific sMtCK isoform has been shown to be more
sensitive than the ubiquitous uMtCK with respect to different
molecular properties of the enzyme [6,24,30]. Therefore, we
have made an attempt to compare the DXR effects on Cr-
stimulated respiration observed in the heart with those present
also in brain mitochondria. Incubation of isolated rat brain
mitochondria was performed both at RTand on ice to exclude an
effect of incubation temperature on mitochondrial function, but
no major difference was observed. In the absence of Cr, DXR
showed a clear inhibitory effect on ADP-stimulated respiration,
similar to that seen with heart mitochondria (Fig. 6A). In the
the steady-state phase (see arrows in Fig. 6A). These data
revealed a much lower or even no deleterious impact of DXR on
Cr-stimulated respiration in brain mitochondria (Fig. 6B).
This could be due to the preservation of the uMtCK-ANT
microcompartment in the brain mitochondria, which is sup-
ported by the only marginal detachment of membrane-bound
uMtCKofbrain mitochondria byDXR (Fig.7B)andalso byour
earlier in vitro study . Similarly as in the heart, uMtCK
enzymaticactivity remained unchangedduring1hofincubation
with DXR (Fig. 7A). In some experiments, such an incubation
slightly increased state 4 respiration as measured after inhibition
Fig. 5. Effect of DXR on enzymatic activity and membrane binding of sMtCK in
heart mitochondria. sMtCK-specific activity in bovine heart mitochondria
exposed to different DXR concentrations during 1 h at RT (A) and during 24 h at
4 °C (B); in panel B, mitochondria were preincubated without (control, CTR) or
with DXR in absence (white bars) or in presence of 15 mM reduced glutathione
(GSH; hatched bars). Control sMtCK activity in bovine heart mitochondria was
1.3±0.3 U/mg protein (n=3). (C) sMtCK probed by immunoblotting in total
control and DXR-treated rat mitochondria (100 μM DXR pro 1 mg/ml
mitochondrial protein during 1 h at RT) and in corresponding supernatants from
the same frozen–thawed mitochondria.
1281 M. Tokarska-Schlattner et al. / Biochimica et Biophysica Acta 1767 (2007) 1276–1284
of ANT by atractyloside, possibly due to the partial permeabi-
lization of inner membrane. Importantly, after longer incubation
times (4 h with 100 μM DXR/mg protein), residual CK activity
9% of control), which would again argue for the better
preservation of the functional integrity of the uMtCK-ANT
3.5. Concluding remarks
mitochondria are not straightforward, since the mitochondria
from different tissue differ in many respects, as membrane
permeability for substrates and ADP, apparent Kmfor ADP of
ANT, and repartition of respiratory control exerted by different
elements of respiratory chain. Moreover, it is to note that
treatment of isolated mitochondria with DXR allowed us to
analyze acute effects of the drug. One can expect that chronic
exposure to DXR in vivo will induce progression of substantial
direct and radical-mediated molecular damage of MtCK (see
), which will further compromise MtCK functions, includ-
ing energy channeling and signaling between mitochondria and
cytosol [3,5], regulation of mitochondrial respiration, formation
of the contact sites , Mt-CK-mediated inner and outer
mitochondrial membrane cross-linking , MtCK-mediated
lipid exchange between inner and outer membrane  and
finally prevention of radical generation  and opening of the
mitochondrial permeability pore , also termed “mitochon-
Fig. 6. Effect of DXR on Cr-stimulated respiration in rat brain mitochondria. (A) Representative traces of kinetics of oxygen consumption in control and DXR-treated
Fig. 7. Effect of DXR on enzymatic activity and membrane binding of uMtCK in
without(control)andwith 100 μM DXR pro1 mg/mlmitochondrialprotein during
1 h at RT (n=3). Absolute values of control uMtCK activity were 2.3±0.3 U/mg
protein for mitochondria kept at RT in comparison to 2.0±0.4 U/mg for
mitochondria kept on ice. (B) uMtCK probed by immunoblotting in total control
and DXR-treated mitochondria and in corresponding supernatants from the same
frozen–thawed mitochondria from rat brain (condition of incubation as in panel A).
1282M. Tokarska-Schlattner et al. / Biochimica et Biophysica Acta 1767 (2007) 1276–1284
drial permeability transition”, which has been observed as a
result of DXR treatment in different cardiotoxicity models .
In conclusion, our study clearly shows that DXR affects
primarily Cr-stimulated respiration and that if reduction in Cr-
stimulated respiration is not taken into account, inhibition of
mitochondrial function by DXR is largely underestimated. This
effect would be particularly marked in tissues expressing
sMtCK, as heart and skeletal muscles. The disturbance of reg-
ulation of mitochondrial respiration by MtCK can be very
harmful at elevated work and can contribute to reduced exercise
tolerance, affecting up to 70% of cancer patients during and
after therapy .
We thank Drs. Dieter Brdiczka and Gregory Brewer for their
helpful suggestions, all members of our groups for the
stimulating discussions, as well Dr. Valdur Saks for a critical
reading of the manuscript. Mrs. Magdalena Livingstone and Dr.
Richard Munton are acknowledged for a gift of anti-PSD95
antibody. This work was supported by grants of Schweizerische
Herzstiftung (to T.W., M.T.-S. and U.S.), Wolfermann-Nägeli-
Stiftung (to U.S. and T.W.), Schweizer Krebsliga (to T.W. and
U.S.), Zentralschweizer Krebsstiftung (to U.S. and T.W.),
Zürcher Krebsliga (to U.S. and M.T.-S.), and Novartis Stiftung
für medizinisch-biologische Forschung (to U.S.).
 M. Tokarska-Schlattner, M. Zaugg, C. Zuppinger, T. Wallimann, U.
Schlattner, New insights into doxorubicin-induced cardiotoxicity: the
critical role of cellular energetics, J. Mol. Cell. Cardiol. 41 (2006)
 V.A. Saks, R. Ventura-Clapier, M.K. Aliev, Metabolic control and metabolic
capacity: two aspects of creatine kinase functioning in the cells, Biochim.
Biophys. Acta 1274 (1996) 81–88.
 U. Schlattner, M. Tokarska-Schlattner, T. Wallimann, Mitochondrial
creatine kinase in human health and disease, Biochim. Biophys. Acta
1762 (2006) 164–180.
 U. Schlattner, T. Wallimann, in: W.J. Lennarz, M.D. Lane (Eds.),
Encyclopedia of Biological Chemistry, Academic Press, New York, USA,
2004, pp. 646–651.
 V. Saks, P. Dzeja, U. Schlattner, M. Vendelin, A. Terzic, T. Wallimann,
Cardiac system bioenergetics: metabolic basis of the Frank-Starling law,
J. Physiol. 571 (2006) 253–273.
 M. Tokarska-Schlattner, T. Wallimann, U. Schlattner, Multiple interference
of anthracyclines with mitochondrial creatine kinases: preferential damage
of the cardiac isoenzyme and its implications for drug cardiotoxicity, Mol.
Pharmacol. 61 (2002) 516–523.
 R.F. Epand, M. Tokarska-Schlattner, U. Schlattner, T. Wallimann, R.M.
Epand, Cardiolipin clusters and membrane domain formation induced by
mitochondrial proteins, J. Mol. Biol. 365 (2007) 968–980.
 D. Brdiczka, G. Beutner, A. Ruck, M. Dolder, T. Wallimann, The molecular
structure of mitochondrial contact sites. Their role in regulation of energy
metabolism and permeability transition, Biofactors 8 (1998) 235–242.
 M. Tokarska-Schlattner, M. Zaugg, R. da Silva, E. Lucchinetti, M.C.
Schaub, T. Wallimann, U. Schlattner, Acute toxicity of doxorubicin on
isolated perfused heart: response of kinases regulating energy supply, Am.
J. Physiol. Heart Circ. Physiol. 289 (2005) H37–H47.
 M. Wyss, J. Smeitink, R.A. Wevers, T. Wallimann, Mitochondrial creatine
kinase: a key enzyme of aerobic energy metabolism, Biochim. Biophys.
Acta 1102 (1992) 119–166.
 W.E. Jacobus, Respiratory control and the integration of heart high-energy
phosphate metabolism by mitochondrial creatine kinase, Annu. Rev.
Physiol. 47 (1985) 707–725.
 A. Boveris, N. Oshino, B. Chance, The cellular production of hydrogen
peroxide, Biochem. J. 128 (1972) 617–630.
Holub, M.F. Oliveira, A. Galina, Mitochondrial creatine kinase activity
prevents reactive oxygen species generation: antioxidant role of mitochon-
drial kinase-dependent ADP re-cycling activity, J. Biol. Chem. 281 (2006)
 M. Dolder, B. Walzel, O. Speer, U. Schlattner, T. Wallimann, Inhibition of
the mitochondrial permeability transition by creatine kinase substrates.
Requirement for microcompartmentation, J. Biol. Chem. 278 (2003)
 D. Cheneval, E. Carafoli, G.L. Powell, D. Marsh, A spin-label electron
spin resonance study of the binding of mitochondrial creatine kinase to
cardiolipin, Eur. J. Biochem. 186 (1989) 415–419.
 D. Cheneval, M. Muller, R. Toni, S. Ruetz, E. Carafoli, Adriamycin as a
probefor thetransversaldistributionof cardiolipinintheinnermitochondrial
membrane, J. Biol. Chem. 260 (1985) 13003–13007.
 M.J. Vacheron, E. Clottes, C. Chautard, C. Vial, Mitochondrial creatine
kinase interaction with phospholipid vesicles, Arch. Biochem. Biophys.
344 (1997) 316–324.
 G. Minotti, P. Menna, E. Salvatorelli, G. Cairo, L. Gianni, Anthracyclines:
molecularadvances and pharmacologic developments in antitumor activity
and cardiotoxicity, Pharmacol. Rev. 56 (2004) 185–229.
 M. Dolder, S. Wendt, T. Wallimann, Mitochondrial creatine kinase in
Recept. 10 (2001) 93–111.
 P. Koufen, A. Ruck, D. Brdiczka, S. Wendt, T. Wallimann, G. Stark, Free
radical-induced inactivation of creatine kinase: influence on the octameric
and dimeric states of the mitochondrial enzyme (Mib-CK), Biochem. J.
344 (Pt 2) (1999) 413–417.
 M.J. Mihm, F. Yu, D.M. Weinstein, P.J. Reiser, J.A. Bauer, Intracellular
distribution of peroxynitrite during doxorubicin cardiomyopathy: evidence
for selective impairment of myofibrillar creatine kinase, Br. J. Pharmacol.
135 (2002) 581–588.
 S. Soboll, D. Brdiczka, D. Jahnke, A. Schmidt, U. Schlattner, S. Wendt, M.
Wyss, T. Wallimann, Octamer–dimer transitions of mitochondrial creatine
kinase in heart disease, J. Mol. Cell. Cardiol. 31 (1999) 857–866.
 O. Stachowiak, M. Dolder, T. Wallimann, C. Richter, Mitochondrial
creatine kinase is a prime target of peroxynitrite-induced modification and
inactivation, J. Biol. Chem. 273 (1998) 16694–16699.
 S. Wendt, U. Schlattner, T. Wallimann, Differential effects of peroxyni-
trite on human mitochondrial creatine kinase isoenzymes. Inactivation,
octamer destabilization, and identification of involved residues, J. Biol.
Chem. 278 (2003) 1125–1130.
 A.I. Toleikis, A.A. Kal'venas, P.P. Dzheia, A.K. Prashkiavichius, A.A.
Iasaitis, Functional changes in the mitochondrial site of adenylate kinase
and creatine kinase systems of energy transport induced by myocardial
ischemia and adriablastin, Biokhimiia 53 (1988) 649–654.
 W.E. Jacobus, A.L. Lehninger, Creatine kinase of rat heart mitochondria.
Coupling of creatine phosphorylation to electron transport, J. Biol. Chem.
248 (1973) 4803–4810.
 U. Schlattner, M. Eder, M. Dolder, Z.A. Khuchua, A.W. Strauss, T.
Wallimann, Divergent enzyme kinetics and structural properties of the two
human mitochondrial creatine kinase isoenzymes, Biol. Chem. 381 (2000)
 M. Wyss, J. Schlegel, P. James, H.M. Eppenberger, T. Wallimann,
Mitochondrial creatine kinase from chicken brain. Purification, biophysi-
cal characterization, and generation of heterodimeric and heterooctameric
molecules with subunits of other creatine kinase isoenzymes, J. Biol.
Chem. 265 (1990) 15900–15908.
 U. Schlattner, N. Mockli, O. Speer, S. Werner, T. Wallimann, Creatine
kinase and creatine transporter in normal, wounded, and diseased skin,
J. Invest. Dermatol. 118 (2002) 416–423.
 U. Schlattner, T. Wallimann, Octamers of mitochondrial creatine kinase
1283M. Tokarska-Schlattner et al. / Biochimica et Biophysica Acta 1767 (2007) 1276–1284
 M. Gosalvez, M. Blanco, J. Hunter, M. Miko, B. Chance, Effects of
anticancer agents on the respiration of isolated mitochondria and tumor
cells, Eur. J. Cancer 10 (1974) 567–574.
 H. Muhammed, T. Ramasarma, C.K. Kurup, Inhibition of mitochon-
drial oxidative phosphorylation by adriamycin, Biochim. Biophys. Acta
722 (1983) 43–50.
 C. Bianchi, A. Bagnato, M.G. Paggi, A. Floridi, Effect of adriamycin on
electron transport in rat heart, liver, and tumor mitochondria, Exp. Mol.
Pathol. 46 (1987) 123–135.
 D.A. Gewirtz, A critical evaluation of the mechanisms of action proposed
for the antitumor effects of the anthracycline antibiotics adriamycin and
daunorubicin, Biochem. Pharmacol. 57 (1999) 727–741.
 V. Saks (Ed.), Molecular Systems Bioenergetics—Energy for Life, Wiley-
VCH, Weinheim, 2007.
 V. Saks, M. Aliev, R. Guzun, N. Beraud, C. Monge, T. Anmann, A.V.
Kuznetsov, E. Seppet, Biophysics of the organized metabolic networks in
muscle and brain cells, Recent Res. Devel. Biophysics 5 (2006) 1–49.
 R. Ventura-Clapier, A. Kuznetsov, V. Veksler, E. Boehm, K. Anflous,
Functional coupling of creatine kinases in muscles: species and tissue
specificity, Mol. Cell. Biochem. 184 (1998) 231–247.
 K. Anflous, V. Veksler, P. Mateo, F. Samson, V. Saks, R. Ventura-Clapier,
Mitochondrial creatine kinase isoform expression does not correlate with
its mode of action, Biochem. J. 322 (Pt 1) (1997) 73–78.
 Z.A. Khuchua, W. Qin, J. Boero, J. Cheng, R.M. Payne, V.A. Saks, A.W.
Strauss, Octamer formation and coupling of cardiac sarcomeric mitochon-
drial creatine kinase are mediated by charged N-terminal residues, J. Biol.
Chem. 273 (1998) 22990–22996.
 P.C. Pelikan, G. Gerstenblith, K. Vandegaer, W.E. Jacobus, Absence of
acute doxorubicin-induced dysfunction of heart mitochondrial oxidative
phosphorylation and creatine kinase activities, Proc. Soc. Exp. Biol. Med.
188 (1988) 7–16.
 T. Miura, S. Muraoka, Y. Fujimoto, Inactivation of creatine kinase by
adriamycin during interaction with horseradish peroxidase, Biochem.
Pharmacol. 60 (2000) 95–99.
 T. Miura, S. Muraoka, T. Ogiso, Adriamycin-Fe(3+)-induced inactivation
of rat heart mitochondrial creatine kinase: sensitivity to lipid peroxidation,
Biol. Pharm. Bull. 17 (1994) 1220–1223.
 H.C. Yen, T.D. Oberley, C.G. Gairola, L.I. Szweda, D.K. St Clair,
Manganese superoxide dismutase protects mitochondrial complex I
against adriamycin-induced cardiomyopathy in transgenic mice, Arch.
Biochem. Biophys. 362 (1999) 59–66.
 O. Speer, N. Back, T. Buerklen, D. Brdiczka, A. Koretsky, T. Wallimann, O.
Eriksson, Octameric mitochondrial creatine kinase induces and stabilizes
contact sites between the inner and outer membrane, Biochem. J. 385 (2005)
 R.F. Epand, U. Schlattner, T. Wallimann, M.L. Lacombe, R.M. Epand,
Novel lipid transfer property of two mitochondrial proteins that bridge the
inner and outer membranes, Biophys. J. 92 (2007) 126–137.
 K.B. Wallace, Doxorubicin-induced cardiac mitochondrionopathy, Phar-
macol. Toxicol. 93 (2003) 105–115.
 R.W. Braith, Role of exercise in rehabilitation of cancer survivors, Pediatr.
Blood Cancer 44 (2005) 595–599.
1284 M. Tokarska-Schlattner et al. / Biochimica et Biophysica Acta 1767 (2007) 1276–1284